Glutathione is a predominant intracellular antioxidant, and is present in CNS cells in millimolar concentrations. (Orlowski M. and Karkowski A., Glutathione metabolism and some possible functions of glutathione in the nervous system. Int Rev Neurobiol 19: 75-121, 1976). Cell turnover of this tripeptide is surprisingly rapid, due at least in part to release of its reduced form (GSH) into the extracellular space. (Yudkoff M., Pleasure D., Cregar L., Lin Z. P., Nissim I., Stem J. and Nissim I., Glutathione turnover in cultured astrocytes: studies with [.sup.15 N]glutamate. J Neurochem 55: 137-145, 1990). GSH release under resting conditions is quantitatively comparable to that of the excitatory neurotransmitters glutamate and aspartate, and is increased by neuronal depolarization via a calcium-dependent process. (Zangerle L., Cuenod M., Winterhalter K. H. and Do K. Q., Screening of thiol compounds: depolarization-induced release of glutathione and cysteine from rat brain slices. J Neurochem 59: 181-189, 1992). The role of extracellular glutathione has not been defined, and has been the subject of considerable conjecture. Until recently, it was presumed to function as an antioxidant and/or an amino acid transporter. GSH alone, without glutathione peroxidase, directly reacts with both the hydroxyl radical and aldehyde products of lipid peroxidation, and may therefore protect cell membranes and associated protein sulfhydryl groups from free radical attack. (Halliwell B. and Gutteridge J. M. C., Free Radicals in Biology and Medicine 2nd ed., pp. 30, 80, 210, Oxford University Press, 1989). .gamma.-glutamyl transpeptidase is primarily located on the outer cell membrane and converts glutathione into gamma-glutamyl amino acids and cysteinylglycine, which are then rapidly transported into cells. (Dringen R., Kranich O., Loschman P. A. and Hamprecht B., Use of dipeptides for the synthesis of glutathione by astroglia-rich primary cultures. J Neurochem 69: 868-74, 1997; Meister A. and Anderson M. E., Glutathione. Annu Rev Biochem 52: 711-760, 1983). Extracellular GSH may therefore serve as a safe carrier molecule for its constituent amino acids (glutamate, cysteine, and glycine), all of which are synaptically active.
A growing body of experimental evidence suggests that extracellular GSH may also specifically interact with membrane receptors on neurons and glia. High affinity, saturable GSH binding sites have been demonstrated in both the rodent and human CNS. (Lanius R. A., Shaw C. A., Wagey R. and Krieger C., Characterization, distribution, and protein kinase C-mediated regulation of [.sup.35 S]glutathione binding sites in mouse and human spinal cord. J Neurochem 63: 155-160, 1994; Ogita K. and Yoneda Y., Temperature-dependent and -independent apparent binding activities of [.sup.3 H]glutathione in brain synaptic membranes. Brain Res 463: 37-46, 1988). Binding is enhanced by protein kinase C activation and is selectively inhibited by cysteine and S-hexylglutathione but not by glutamate. The similar distribution of GSH and glutamate binding sites (Ogita K. and Yoneda Y., Temperature-dependent and -independent apparent binding activities of [.sup.3 H]glutathione in brain synaptic membranes. Brain Res 463: 37-46, 1988) suggests that GSH may act as a neuropeptide at the redox site of the NMDA receptor. GSH potentiated responses to glutamate in cells expressing recombinant NR1-NR2A receptors (Kohr G., Eckardt S., Luddens H., Monyer H. and Seeburg P. H., NMDA receptor channels: subunit-specific potentiation by reducing agents. Neuron 12: 1031-1040, 1994), and increased glutamate and NMDA-induced calcium influx in cultured cerebellar granule cells. (Janaky R., Varga V., Saransaari P. and Oja S. S., Glutathione modulates the N-methyl-D-aspartate (NMDA) receptor-activated calcium influx into cultured rat cerebellar granule cells. Neurosci Lett 156: 153-157, 1993).
Excessive activation of glutamate receptors increases neuronal oxidative stress (Monyer H., Hartley D. M. and Choi D. W., 21-aminosteroids attenuate excitotoxic neuronal injury in cortical cell cultures. Neuron 5: 121-126, 1990; Puttfarcken P. S., Getz R. L. and Coyle J. T., Kainic acid-induced lipid peroxidation: protection with butylated hydroxytoluene and U78517F in primary cultures of cerebellar granule cells. Brain Res 624: 223-232, 1993; Reynolds I. J. and Hastings T., Glutamate induces the production of reactive oxygen species in cultured forebrain neurons following NMDA receptor activation. J Neurosci 15: 3318-3327, 1995) and has been implicated in the pathogenesis of both acute CNS insults and neurodegenerative diseases (Lipton S. A. and Rosenberg P. A., Excitatory amino acids as a final common pathway for neurologic disorders. N Engl J Med 330: 613-622, 1994). While screening antioxidant compounds in a cell culture model of mild excitotoxic injury, marked potentiation of NMDA neurotoxicity by glutathione was unexpectedly observed, consistent with it being a ligand at the NMDA receptor redox site.
The NMDA glutamate receptor may predominate in many disease processes because of the high calcium permeability of its cation channel. One promising method of preventing NMDA receptor overactivation is the pharmacologic modulation of its redox sites. However, since the endogenous ligands at these sites had been unknown, little progress had been reported. Recent results identify glutathione and L-cysteine as endogenous ligands at the redox sites of the NMDA receptor.
The present invention is a method of treating central nervous system diseases and conditions in a mammal, comprising administering a compound that interacts with and modifies one of the redox sites of the NMDA receptor in said mammal. In the present invention, two S-substituted glutathione derivatives S-hexylglutathione and S-methylglutathione, blocked the potentiation of NMDA receptor neurotoxicity produced by reducing agents. Thus, glutathione derivatives provide a novel pharmacologic approach to modulation of NMDA receptor responses.
Present pharmacologic approaches to the NMDA receptor include: 1) competitive antagonists, which are amino acid derivatives that compete with glutamate for receptor binding; 2) noncompetitive antagonists, which block the cation channel; 3) glycine (a co-agonist site antagonist). Unfortunately, these compounds all interfere with normal receptor function. Administration of these drugs often produces psychotic behavior (the street drug PCP is a noncompetitive NMDA antagonist). Also, the ability to make new memory is lost, since the NMDA receptor is crucial to this process.
The present invention facilitates modulation of the redox site to prevent receptor overactivation, but this does not interfere with normal receptor function. Intermediate and long-term administration would therefore be more feasible with the present invention.